PROJECT SUMMARY Lysine acetylation has traditionally been studied as an epigenetic modifier of histone tails within chromatin that provides an important mechanism for regulating gene expression. In the heart, histone acetylation acts as a key regulator of cardiac remodeling and function. However, recent reports have shown that non-histone proteins can be acetylated. Importantly, it has been postulated that the acetylome rivals phosphorylation in prevalence as a post-translational modification. The long-term goal of my lab is to dissect the epigenetic and non-epigenetic actions of lysine acetylation in the regulation of heart failure. The objective of this application is to elucidate the role of acetylation of non-histone proteins in the regulation of cardiac hypertrophy and muscle function, with an emphasis on sarcomeric proteins. Preliminary findings from our lab demonstrate that obesity-mediated cardiac remodeling is associated with significant changes in lysine acetylation of proteins within the left ventricle of mice. Mass spectrometry analyses further demonstrated that, of the 3264 lysine-acetylated non-histone proteins identified, 145 were acetylated on 189 unique acetylation sites, 16 of which were significantly impacted by obesity. Ingenuity Pathway Analysis identified the Cardiovascular Disease Network and revealed LIM domain- binding protein 3 (LDB3) and skeletal muscle alpha actin 1 (ACTA1) as proteins that were significantly impacted by obesity. LDB3 and ACTA1 affect muscle structure, integrity, and cellular motility. In addition, LDB3 has been reported to regulate calcineurin-NFAT signaling pathway, which is important in the development of cardiac hypertrophy. Mutations in LDB3 or ACTA1 have been linked to cardiomyopathies, but whether these proteins are acetylated in the heart remains unknown. This proposal will test the central hypothesis that acetylation of sarcomere proteins, specifically LDB3 and ACTA1, regulate cardiac functions through effects on cardiac hypertrophy and muscle contractility. We have developed three specific aims to test this hypothesis. In Aim 1, we seek to delineate a role for sarcomere protein acetylation in cardiac myocyte function. In Aim 2, we elucidate which proteins regulate sarcomere protein acetylation in cardiac myocytes. And in Aim 3, we use adeno- associated virus serotype 9 (AAV9) to determine the physiological significance of LDB3 and ACTA1 acetylation in vivo. Most studies to date have examined lysine acetylation in the regulation of nucleosomal DNA and gene expression. As such, the proposed research is innovative and will add significant insight into the process of sarcomere protein (non-histone) acetylation in the regulation of cardiac biology.